Genetic polymorphisms and protein expression of NRF2 and ... · 8/9/2012 · 1 Genetic...
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Genetic polymorphisms and protein expression of NRF2 and sulfiredoxin predict
survival outcomes in breast cancer
Jaana M. Hartikainen1-3, Maria Tengström4,5, Veli-Matti Kosma1-3, Vuokko L. Kinnula6, Arto
Mannermaa1-3*, Ylermi Soini1-3*
1Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern
Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
2Biocenter Kuopio and Cancer Center of Eastern Finland, University of Eastern Finland,
P.O. Box 1627, FI-70211 Kuopio, Finland
3Imaging Center, Clinical Pathology, Kuopio University Hospital, P.O. Box 1777, FI-70211
Kuopio, Finland
4Institute of Clinical Medicine, Oncology, University of Eastern Finland, P.O. Box 1627, FI-
70211 Kuopio, Finland
5Cancer Center, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio, Finland
6Department of Medicine, Pulmonary Division, University of Helsinki and Helsinki
University Central Hospital, P.O. Box 440, FI-00014, Helsinki, Finland
*These authors contributed equally to the work.
Running title: NRF2 and sulfiredoxin predict outcome in breast cancer patients
Key words: NRF2, sulfiredoxin, breast, cancer, oxidative stress
Financial support: The Finnish Cancer Society; the Academy of Finland (grant number
127220); EVO Research Fund (grant number 5654113 and 5501) of Kuopio University
Hospital; and the strategic funding of the University of Eastern Finland.
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Corresponding author: Jaana M. Hartikainen, Institute of Clinical Medicine, Pathology
and Forensic Medicine, University of Eastern Finland, Yliopistonranta 1 C, FI-70210
KUOPIO, FINLAND. Phone: +358 40 355 2754. Fax: +358 17 162753. Email:
The authors disclose no potential conflicts of interest.
Word count: 4176 words (excluding affiliations, abstract, references, figure legends and
tables).
Four figures and 3 tables (and 9 supplemental figures and 8 supplemental tables).
Prêcis: Potentially seminal findings identify biomarkers in a core ROS stress pathway that
may be generally impactful in determining the survival outcomes of patients with breast
cancer.
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ABSTRACT
NRF2 activates several protective genes, such as sulfiredoxin (SRXN1), as a response to
oxidative and xenobiotic stress. Defects in NRF2 pathway may increase cancer
susceptibility. In tumor cells activation of NRF2 may lead to chemo- and radioresistance
and thus affect patient outcome. Nine single nucleotide polymorphisms (SNPs) on NRF2
gene and eight on SRXN1 were genotyped in 452 breast cancer patients and 370 controls.
Protein expression of NRF2 and SRXN1 was studied in 373 breast carcinomas by
immunohistochemistry. Statistical significance of the associations between genotypes,
protein expression, clinicopathological variables and survival was assessed. A high level
(>25%) of cytoplasmic NRF2 positivity was observed in 237/361 (66%) and SRXN1
positivity was observed in 82/363 (23%) of the cases. The NRF2 rs6721961 genotype TT
was associated with increased risk of breast cancer (P=0.008, OR=4.656, CI=1.350-
16.063) and the T allele was associated with a low extent of NRF2 protein expression
(P=0.0003, OR=2.420, CI=1.491-3.926) and negative SRXN1 expression (P=0.047,
OR=1.867, CI=1.002-3.478). The NRF2 rs2886162 allele A was associated with low NRF2
expression (P=0.011, OR=1.988, CI=1.162-3.400) and the AA genotype was associated
with a worse survival (P=0.032, HR=1.687, CI=1.047-2.748). The NRF2 rs1962142 T
allele was associated with a low level of cytoplasmic NRF2 expression (P=0.036) and
negative sulfiredoxin expression (P=0.042). The NRF2 rs2706110 AA genotype was
associated with an increased risk of breast cancer and the SRXN1 rs6053666 C allele was
associated with a decrease in breast cancer risk (P values 0.011 and 0.017). NRF2 and
SRXN1 genetic polymorphisms are associated with breast cancer risk and survival,
implicating that mechanisms associated with ROS and NRF2 pathway are involved in
breast cancer initiation and progression.
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INTRODUCTION
Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcriptional factor which senses
oxidative and xenobiotic stress in the cells (1). When such stress occurs, NRF2 is released
from a complex formed with KEAP1 (Kelch-like ECH-associated protein 1), a substrate
adaptor of a Cullin 3-based E3 ubiquitin ligase complex, and moves to the nucleus where it
associates with maf proteins and then upregulates several stress related genes such as
glutathione-S-transferases, thioredoxin, thioredoxin reductases, peroxiredoxins, gamma
glutamyl cysteine ligase, heme oxygenase 1, NADPH kinone oxidoreductase and
multidrug resistance genes (1, 2). If NRF2 stays in a complex with KEAP1 in cytoplasm, it
is degraded through the proteasome pathway (1, 2).
The function of NRF2 is important in the pathogenesis of several diseases. Mice with a
non-functioning Nrf2 gene develop early onset emphysema because of a deficient
antioxidative response (3). Similarly mice lacking Nrf2 develop nutritional steatohepatitis
(4). A high amount of unbound NRF2 in cancer cells results in chemoresistance of the
tumor cells (5). The importance of NRF2 and KEAP1 in tumorigenesis is underlined by the
fact that tumor cells may contain mutations in the respective genes. NRF2 mutations are
present in oesophageal, skin, larynx and lung cancer with a 6-13 % frequency with the
highest prevalence in squamous cell carcinomas (6). A similar mutational frequency has
been found for KEAP1 with an incidence of 15 % in lung cancer (7). Loss of KEAP1
function leads to increased NRF2 concentration and chemoresistancy in non-small cell
lung cancer (8). In addition to somatic mutations, genomic low penetrance DNA variations
could have an effect to the function of these genes and their downstream targets.
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Peroxiredoxins are enzymes, which have capability in scavenging hydrogen peroxide and
other peroxides (9, 10). Peroxiredoxins may undergo reversible oxidation in their cysteine
sites to sulfinic acid rendering the molecules to degradation (10, 11). Sulfiredoxin
catalyses the reversal of overoxidation of peroxiredoxins, thus salvaging them from
inactivation (11). Sulfiredoxin is also involved in deglutathionylation of proteins following
nitrosactive or oxidative stress (12). In cell lines, overexpression of sulfiredoxin has been
shown to stimulate cell proliferation and apoptosis induced by cisplatin, the effects of
which were mediated by phosphorylation of cell cycle regulators and kinases (13).
Sulfiredoxin is induced by NRF2 and AP-1 and protects the lung from tobacco mediated
oxidative damage (14, 15). Increased sulfiredoxin has been linked with oncogenic
transformation, and it is overexpressed in various skin cancers (16).
There are many risk factors associated with the development of breast carcinoma, and 1-5
% of them have a hereditary basis (17). Even though oxidative damage is considered as
one mechanism for cancer development, its role in breast cancer has not been extensively
studied. Since NRF2 is known to be a sensor of oxidative damage, we studied its
expression in different types of breast carcinoma. Additionally we investigated the
expression and significance of sulfiredoxin, a known target for NRF2, in breast carcinoma.
We also evaluated the effect of NRF2 and SRXN1 genetic variation on the protein
expression, as well as their role in breast cancer risk and development. The genetic
variants and the level of protein staining were also evaluated as predictive factors. This is
the first report on SRXN1 polymorphisms in (breast) cancer.
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MATERIALS AND METHODS
DNA samples
DNA from 452 patients with invasive breast cancer and 370 control subjects from the
Kuopio Breast Cancer Project (KBCP) sample set were available for genotyping
(Supplemental Table S1). The KBCP sample set consists of 497 prospective breast cancer
cases and 458 controls from the province of Northern Savo in Eastern Finland. The KBCP
sample material is characterized in more detail by Hartikainen et al. 2005 (18) and
Pellikainen et al. 2003 (19). Genomic DNA was extracted from peripheral blood
lymphocytes of both cases and controls using standard procedures (20). The KBCP has
been approved by the ethical committee of University of Eastern Finland and Kuopio
University Hospital.
Tumor material in tissue microarray
The tumor material consisted of 373 cases of invasive breast carcinomas included in the
KBCP. The clinical characteristics of the material are shown in Supplemental Table S1.
Paraffin-embedded tumor tissue from the primary tumor was obtained from breast cancer
surgery. Tissue microarray was constructed as previously described (21).
Single nucleotide polymorphism (SNP) selection
Tagging single nucleotide polymorphism (tagSNPs) for NRF2 and SRXN1 genes were
selected using the HapMap Genome Browser release 2 (Phase 3, NCBI build 36, bdSNP
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b126) as of February 24th and November 8th 2010 (Ref. 22). TagSNPs for regions
chr2:177799989-177853228 and chr20:573580-583579 were picked out for the CEU
population using the Tagger multimarker algorithm with r2 cutoff at 0.8 and MAF (minor
allele frequency) cutoff at 0.05. Two functional polymorphisms on the NRF2 promoter
region were selected on the basis of previous publications (23, 24). (Supplemental Figure
S1A and B)
Genotyping of NRF2 and SRXN1 SNPs
Genotyping of six NRF2 and eight SRXN1 tagging single nucleotide polymorphisms
(TagSNPs) and two NRF2 functional SNPs was done using MassARRAY® (Sequenom
Inc., San Diego, CA, USA) and iPLEX® Gold (Sequenom Inc.) on 384-well plate format.
MassARRAY mass spectrometer (Sequenom Inc.) was used for spectra acquisitions from
the SpetroCHIP. Data analysis and genotype calling were done using TyperAnalyzer
Software version 4.0.3.18 (Sequenom Inc.). Each 384-well plate contained a minimum of 8
non template controls. Duplicate analysis was done for 6.7% of the samples for quality
control. All primer sequences and reaction conditions are available upon request.
Genotyping of the NRF2 tagSNP rs2886162 was performed by 5’ nuclease assay
(TaqMan) using the Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA)
according to manufacturer’s instructions. Primers and probes for rs2886162 were supplied
from Applied Biosystems (Foster City, CA, USA) as TaqMan Genotyping Assays.
Reactions were carried out in 10 μl volume in 96-well format as previously described (21).
Duplicate genotypes were done for 4.2 % of samples for quality control. If the duplicate
and its pair were discordant the genotypes for the sample would be discarded.
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Immunohistochemistry
Four-�m-thick tissue sections were cut from the paraffin-embedded blocks. The
construction of the microarray blocks and the immunohistochemical staining procedure
has been described previously (21, 25). The primary antibodies, rabbit polyclonal anti-
human NRF2 (sc-722, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and rabbit
polyclonal anti-human sulfiredoxin (14273-1-AP, Protein Tech Group, Chicago, IL, USA)
were diluted with 1 % bovine serum albumin in PBS to 1:200 and 1:500 working solutions,
respectively. The evaluation of NRF2 immunostaining was performed separately in tumor
cell nuclei and cytoplasm. For sulfiredoxin cytoplasmic immunoreactivity was evaluated.
The results for NRF2 were semiquantitated as follows; 0-5 %=negative, over 5 to 25 %=
weak positivity, over 25 to 75 %=moderate positivity, over 75 to 100 %= strong positivity.
In the analyses, NRF2 expression was divided in two groups, low extent (< 25 %) and high
extent (> 25 %) expression. For sulfiredoxin the presence (> 1 %) or absence of
cytoplasmic expression was recorded.
Statistical analysis
The statistical analyses were performed with SPSS for Windows software v 14.0 (SPSS,
Chicago, IL,USA). Continuous data were compared using analysis of variance (ANOVA).
When ANOVA results indicated that groups differed, post hoc comparisons were
performed using two-tailed t-tests. Categorical data were compared using Fisher’s exact
test designed for small sample groups. The significance levels for comparisons of the
genotype frequencies between cases and controls, and for the association between the
genotypes and protein expression and clinical variables (tumor grade and size, histological
type, nodal status, estrogen receptor status, progesterone receptor status, HER status)
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among the cases were also computed using Armitage's trend test. The concordance of the
genotypes with Hardy-Weinberg equilibrium was tested using standard chi-squared test.
Survival data were analyzed using the Kaplan-Meier method with the use of the log-rank,
Breslow and Tarone-Ware test in SPSS v 14.0 (SPSS Inc.). In multivariate survival
analyses the Cox regression analysis in SPSS v 14.0 (SPSS Inc.) was used. P-values less
than 0.05 (two-sided) were considered statistically significant in all tests.
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RESULTS
NRF2 rs6721961 and rs2706110 associate with increased risk of breast cancer and
SRXN1 rs6053666 protects against breast cancer
Seven tagging SNPs (rs1806649, rs2886162, rs1962142, rs2364722, rs10183914,
rs2706110 and rs13035806) and two functional SNPs (rs6721961 and rs6706649) were
analysed in the NRF2 gene region. Eight tagging SNPs (rs6085283, rs13043781,
rs6076869, rs6053666, rs2008022, rs6116929, rs7269823 and rs6053728) were analyzed
in the SRXN1 gene region (Supplemental Table S2). The SNP genotypes were tested for
concordance with the Hardy-Weinberg equilibrium (HWE). Among the controls NRF2
rs6706649 deviated slightly from HWE with a P value of 0.029. All other genotypes were
concordant with the HWE.
Among the invasive breast cancer cases an association with breast cancer risk was
observed with NRF2 rs6721961 and rs2706110, and SRXN1 rs6053666 genotypes (Table
1). The rare homozygous genotypes of NRF2 rs6721961 (TT) and rs2706110 (AA)
associated with increased risk of breast cancer, whereas the common allele was protective
(Table 1). The rare allele C of SRXN1 rs6053666 was protective (Table 1). A near
significant association was observed with NRF2 rs13035806 (Table 1).
NRF2 expression associates with sulfiredoxin expression
High extent (> 25 %) cytoplasmic NRF2 positivity was seen in 66 % (237/361) and nuclear
(> 25 %) positivity in 26 % (96/365) of cases (Figure 1A-D). High extent nuclear positivity
was observed in 20 % (43/219) of ductal, 47 % (33/70) of lobular and 29 % (17/59) of other
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types. Most notably, lobular carcinomas showed significantly more high extent nuclear
NRF2 expression than ductal ones (P=0.001). Twenty-three percent (82/363) of the breast
tumors displayed positivity for sulfiredoxin (Figure 1E and F). Twenty-three percent
(50/219) of ductal, 15 % (10/68) of lobular and 30 % (15/50) of other types expressed
positivity. Nuclear and cytoplasmic NRF2 expression was associated with sulfiredoxin
expression (P=0.003 and P=0.008, respectively, Supplemental Table S3).
NRF2 SNP rare alleles associate with low extent cytoplasmic NRF2 and sulfiredoxin
protein expression
A significant association was observed with cytoplasmic NRF2 protein expression and the
NRF2 rs1962142, rs2886162 and rs6721961 genotypes among the invasive breast cancer
cases, the rare alleles associating with low extent cytoplasmic NRF2 protein expression
(Table 2). The rare alleles of NRF2 rs1962142 and rs6721961 also associated with
negative sulfiredoxin protein expression (Table 2). More specifically, NRF2 rs6721961 rare
allele associated with grade 2 tumors (P(Overall)=0.041, P(Allele specific)=0.012, OR=1.975,
CI=1.159-3.365) and NRF2 rs2886162 rare homozygous genotype AA associated with ER
positive breast cancer (P(Overall)=0.008, P(Allele specific)=0.008, OR=2.518, CI=1.276-4.969).
NRF2 SNP rs1962142 allele T also associated with grade 2 tumors (data not shown). This
SNP resides in the same haplotype block with rs6721961 and most likely represents the
same association as rs6721961.
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SRXN1 SNP rs6076869 rare allele associates with cytoplasmic NRF2 protein
expression
SRXN1 rs6076869 genotypes associated with cytoplasmic NRF2 protein expression
among the invasive breast cancer cases. The rare allele T associated with high extent
cytoplasmic NRF2 protein expression (Table 2) and with lobular histology (P(Overall)=0.019,
P(Allele specific)=0.022, OR=1.830, CI=1.092-3.066).
NRF2 and SRXN1 genotypes associate with prognosis/survival
NRF2 rs2886162 rare homozygous genotype AA associated with a worse survival
compared to the carriers of the common allele G (Kaplan-Meier P(log rank)=0.017,
Supplemental Table S4, Supplemental Figure S2). This association remained significant in
the multivariate analysis (Cox regression P=0.032, HR=1.687, CI=1.047-2.748, Table 3,
Figure 2). In this multivariate analysis also the cytoplasmic NRF2 expression was included
but it did not associate with survival. However, in the Kaplan-Meier analysis a difference in
the genotype-associated survival was observed between the strata (cytoplasmic NRF2 low
extent vs. high extent, P=0.023, log rank 5.163), implying that the genotypes association is
most significant among those with low extent cytoplasmic NRF2 only (Supplemental Table
S5, Supplemental Figure S3A and B). Similar trend was observed with nuclear NRF2
protein expression (P=0.019, log rank 5.490) (Supplemental Table S5, Supplemental
Figure S4A and B).
SRXN1 rs6116929 rare homozygous genotype GG and rs2008022 rare allele carriers
CA&AA had better survival compared to the common allele (P(log rank)=0.063 and P(log
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rank)=0.012, respectively, Supplemental Table S4, Supplemental Figure S5A and B).
SRXN1 rs7269823 and rs6085283 rare allele carriers (AG&GG and CT&TT, respectively)
had poorer survival compared to the common homozygous genotype (P(log rank)=0.030 and
P(log rank)=0.015, respectively, Supplemental Table S4, Supplemental Figure S5C and D). In
the Cox regression analysis including all four survival-associated SRXN1 polymorphisms
only rs2008022 remained significant (P=0.012, HR=1.645, CI=1.116-2.425). None of these
four polymorphisms however, were independently significant prognostic factors in the
multivariate analysis including tumor grade, nodal status, ER status, PR status, histological
type, tumor size and HER2 status (Cox regression, data not shown).
Effect of combined NRF2 and SRXN1 genotypes on prognosis/survival
We further studied the effect of the combined SRXN1 survival-associated polymorphisms
by summing up the number of the risk alleles of the SRXN1 polymorphisms rs61169295,
rs2008022, rs7269823 and rs6085283 for each patient. The highest value possible was 8
and the lowest was zero. The patients were divided in two groups; 0-3 risk alleles and 4-8
risk alleles. A trend towards poorer survival was observed with increasing amount of risk
alleles in Kaplan-Meier analysis (P(log rank)=0.009 for 0-3 vs. 4-8 risk alleles, Supplemental
Table S4 Supplemental Figure S6). However, the poorest survival was still defined by
rs2008022. When the effect of NRF2 rs2886162 was also considered together with the
combined SRXN1 SNPs there was a difference in the survival between the strata defined
by rs2886162 genotype (P=0.014, log rank 6.009). Among the rs2886162 rare allele (A)
carriers poorer survival was observed among those with 4-8 SRXN1 risk alleles(P(log
rank)=0.010) but no difference among the rs2886162 common homozygotes (GG) was
observed (P(log rank)=0.638) (Supplemental Figure S7A and B). This reflects that the
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rs2886162 genotype is a stronger prognostic factor than the combined SRXN1 SNP
genotypes; otherwise the effect on survival by SRXN1 genotypes should also be seen
among the rs2886162 common homozygotes. Indeed, in the multivariate analysis
including the combined SRXN1 genotypes, NRF2 rs2886162 genotype and other
prognostic factors, only rs2886162 genotype, nodal status and HER2 status remained
significant (Supplemental Table S6, Supplemental Figure S8A). Similar results were
obtained from the multivariate analysis including the clinicopathological variables, NRF2
protein expression (cytoplasmic and nuclear), sulfiredoxin protein expression, combined
SRXN1 genotypes and rs2886162 (Supplemental Table S7, Supplemental Figure S8B).
NRF2 rs2886162 AA genotype independently predicts poorer survival among
patients who received chemotherapy or radiation therapy
The effect of NRF2 rs2886162 rare homozygous genotype AA on poor prognosis was also
seen separately in the group that had received adjuvant chemotherapy (CT) and among
those that received postoperative radiation therapy (RT). In the group that had received
adjuvant CT the rs2886162 genotype AA associated with poorer breast cancer survival
(P=0.019, HR=2.43, CI=1.16-5.08) (Figure 3A) and with poorer recurrence-free survival
(P=0.003, HR=2.83, CI=1.43-5.61). In the group that received postoperative RT the
rs2886162 genotype AA associated with poorer recurrence-free survival (P=0.025,
HR=1.68, CI=1.07-2.64) (Figure 3B). Among patients who did not receive any adjuvant
therapy (n=137) the rs2886162 genotypes did not associate with survival (data not
shown).
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SRXN1 genotypes independently predict survival among patients receiving
radiation treatment
The effect of the SRXN1 genotypes on prognosis also holds when radiation treatment is
taken into account. SRXN1 rs6116929 rare homozygous genotype GG and rs2008022
rare allele carriers CA&AA predicted better prognosis among the patients who received RT
(Supplemental Table S8, Figure 4A and B). Also, among the patients treated with RT, the
SRXN1 rs7269823 and rs6085283 rare allele carriers (AG&GG and CT&TT, respectively)
had poorer survival compared to the patients carrying the common homozygous
genotypes (Supplemental Table S8, Figure 4C and D). In addition, among the patients
treated with RT the SRXN1 rs6053666 rare homozygous genotype CC predicted better
prognosis compared to the common allele carriers (Supplemental Table S8). Interestingly,
rs6053666 rare allele also associates with decreased breast cancer risk.
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DISCUSSION
NRF2 is a transcription factor which senses xenobiotic and oxidative stress and activates
several antioxidative and other protective genes if such stress occurs. Upregulation of
NRF2 may thus protect cells from oxidative damage and prevent initiation of
carcinogenesis due to mutations caused by such damage. In tumor tissue, on the other
hand, activation of NRF2 leads to increased chemo- and radioresistance of the tumor cells
which is reflected by the fact that many tumors display elevated levels of antioxidative
enzymes compared to normal tissues (26). Recent findings suggest that enhanced
detoxification of reactive oxygen species with additional NRF2 functions may in fact be
also protumorigenic (27).
Our results demonstrate that in the NRF2 pathway there are genetic polymorphisms that
affect both the susceptibility for breast cancer and the outcome of the breast cancer
patients, thus underlining the complex effect of NRF2 in cancer progression. On one hand,
the NRF2 promoter polymorphism rs6721961 associates with breast cancer risk referring
to the role of NRF2 in cancer predisposition. The rare allele T associates with increased
risk of breast cancer and low protein level of both NRF2 and sulfiredoxin. The T allele is
predicted to destroy a binding site for a transcription factor (intronic enhancer) c-Rel
(FastSNP, ref. 28) (Supplemental Table S9). Previously, rs6721961 has been shown to be
functional (24) and hence it would directly affect the protein expression level of NRF2.
Indeed, here we have demonstrated the connection between the T allele and decreased
NRF2 protein expression in breast cancer tissue, as well as the resulting decrease in
sulfiredoxin expression. This is also concordant with the hypothesis that impaired NRF2
function leads to decreased sulfiredoxin function, which in turn affects the function of
peroxiredoxins and leads to increased cancer proneness. Decreased NRF2 level
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presumably might affect also the activation of other NRF2 targets and hence increase
cancer susceptibility.
On the other hand, NRF2 SNP rs2886162 AA genotype associates with low NRF2 protein
expression level and poorer survival. The effect of the genotype on survival was significant
also in the multivariant analysis. In addition, the NRF2 rs2886162 rare homozygous
genotype AA independently predicted poorer survival among patients who received
adjuvant chemotherapy, and the recurrence-free survival was poorer among RT-treated
AA genotype carriers. The rs2886162 association with survival could be through impaired
NRF2 (and sulfiredoxin) function as low cytoplasmic NRF2 may result in low sulfiredoxin,
even though statistically significant association between rs2886162 and negative
sulfiredoxin level was not observed here. (However, a positive overall correlation between
NRF2 expression and sulfiredoxin expression was observed). It is also possible that low
cytoplasmic NRF2 could be explained by the removal of NRF2 from cytoplasm to the
nucleus where it leads to activation of stress response and survival of cancer cells (“NRF2
resistency”) and thus poorer prognosis. The association with poorer survival in this case
could be explained by the fact that SRXN1 is not the sole target for NRF2 activation.
Association of nuclear NRF2 staining with poorer survival has been previously observed in
ovarian carcinoma (29). However, in breast cancer this issue needs further studies,
possibly including also KEAP1.
Previous studies on NRF2 polymorphisms in breast cancer are few. In a cohort of
postmenopausal women, specific polymorphisms on NRF2 (rs1806649), NQO1, NOS3
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and HO-1 did not have any significance for the risk of breast cancer (30). When the risk
polymorphisms of these genes were combined, patients with three risk alleles had a 1.5
fold risk and those with a high iron intake had a greater than 2 fold risk (30). In
postmenopausal women with oral estrogen replacement therapy the NRF2 rs6721961 rare
allele seems to modify the risk of thromboembolism (31). NRF2 polymorphism has,
however, been more extensively studied in pulmonary disease (24, 32, 33). While NRF2
polymorphisms clearly may promote individuals for oxidative damage, no published
studies on their significance in lung cancer exist. Lung cancer, as well known, is
associated with tobacco smoke which among other effects provokes development of ROS
(34). Polymorphisms rs6721961 and rs6706649 have been studied in gastric
carcinogenesis but no overall association with risk was found (35). In gastric cancer,
carcinogenesis is predominantly based on H. pylori induced gastritis leading to gastric
atrophy and cancer, while in breast cancer hormonal factors play a role (36). It is known
that estrogen metabolites induce the formation of reactive oxygen species (37). In this
sense NRF2 and its dysfunction may be more important in breast carcinogenesis than
gastric cancer.
In addition to NRF2 polymorphisms, we observed that polymorphisms on SRXN1 also are
associated with breast cancer risk and survival. Four SRXN1 SNPs associated with breast
cancer survival in Kaplan-Meier analysis (rs6116929, rs2008022, rs7269823 and
rs6085283). Among patients who received RT these SNPs also associated independently
with survival in the multivariate analysis. Interestingly, there are regulatory features in the
regions where rs6116929 and rs6085283 reside (Ensembl, ref. 38). rs6116929 locates in
downstream region and the rare allele G (which associated with better survival) is
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predicted to destroy binding sites for CdxA, cap and deltaE (F-SNP, ref. 39). rs6116929 is
also only 84 base pairs from rs6076869 (D’ 0.99), the rare allele of which associated with
increased NRF2 protein. rs6076869 rare allele T is predicted to destroy a GATA-X binding
site (FastSNP) and to create cap and AP-4 TF binding sites (F-SNP). rs6085283 resides in
intron 1 and the rare allele T (which associated with poorer survival) creates a binding site
for Oct-1 transcription factor (FastSNP). Also, the rs2008022 (in intron 1) rare allele A
(which associated with better survival) destroys a GATA-2 transcription binding site, and is
1079 base pairs from rs13043781 (D’ -0.98), the rare allele of which is predicted to destroy
v-Myb binding site (FastSNP). There were no predicted or detected functional effects for
rs7269823 which also resides in intron 1. However, it is in LD with rs2008022 (D’ -1) and
rs6053666 (D’ 0.7). (Supplemental Table S9). It is possible that these polymorphisms
affect the level or function of sulfiredoxin and the cancer cells exhibiting low sulfiredoxin
expression have lower tolerance for oxidative damage and the response for oxidative
damage is poor, which promotes/enhances the death of the cancer cell and a better
response to treatment and thus, leads to better outcome. The effect of sulfiredoxin in
breast carcinoma could be connected to its role in converting peroxiredoxins to a
functional, reduced state. Some peroxiredoxins have been associated with progression of
breast cancer. Overexpression of peroxiredoxin VI in breast carcinoma cell lines leads to a
more invasive phenotype with a higher proliferative activity (40). Moreover, peroxiredoxin
III promotes breast cancer cell proliferation and peroxiredoxins I, II and III protect cells
from oxidative damage induced apoptosis (41, 42). We also found that SRXN1 rs6053666
rare allele C lowered the risk of breast cancer and the CC genotype associated with better
prognosis among the patients who received RT. Such influences may be ascribed to the
known function of sulfiredoxin on the oxidative state of peroxiredoxins regulating the redox
state and metabolism of hydrogen peroxide in cells. rs6053666 resides on the 3’UTR
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region of the SRXN1 gene and is predicted to participate in splicing regulation (alternative
splicing). Three exonic splicing enhancer (ESE) binding sites are predicted for allele C
(SF2/ASF, SC35 and SRp55), and none for allele T (FastSNP, F-SNP) (Supplemental
Table S9).
The protein expression of NRF2 has not previously been studied in large clinical materials
of breast cancer. Our results show that NRF2 is strongly expressed in the cytoplasm of
breast carcinoma cells, showing a high frequency expression in 66 % of the cases. High
extent nuclear expression, indicating increased functional activity of the protein, was
present in 26 % of the cases. In the histological subgroups, lobular invasive carcinomas
showed a stronger expression of nuclear positivity than ductal ones. Lobular carcinoma is
a tumor type showing low or non-existent expression of E-cadherin. Interestingly, NRF2
activation by sulphoraphane was reported to cause down regulation of EMT type changes
in rat kidney tubular epithelial cells, including E-cadherin (43). Thus, NRF2 activation might
be one additional factor influencing the loss of E-cadherin expression in lobular breast
carcinoma. On the other hand, lobular carcinoma cells could be more sensitive in their
reaction to oxidative stress, leading to a more abundant nuclear expression of NRF2.
Previously, Loignon and co-workers (2009) found that NRF2 protein expression was
decreased in seven of the ten breast cancer cell lines they studied (44). They also
detected lower levels of NRF2 in seven of the ten studied breast cancer tumor samples
compared to normal breast tissue (45). Unfortunately the authors did not specify
subcellular localization of the staining or the histological subgroups of the tumors. High
gene expression of NRF2 has been reported to associate with poor prognosis among ER
positive breast cancer (46).
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To conclude, we have observed that NRF2 and SRXN1 polymorphisms influence breast
cancer susceptibility and survival, and the protein expression has an effect on breast
cancer survival. All in all, ROS associated mechanisms appear to play a role in the
behavior and treatment of breast cancer to the extent of being reflected in the survival of
the patients. Future studies would be needed for the confirmation of the functional SNPs
and their effect on the NRF2 and sulfiredoxin protein expression, as well as studies on
peroxiredoxins and AP-1. Studying antioxidative mechanisms may thus pave the way for
new treatment modalities based on inhibition of such mechanisms in breast cancer cells.
ACKNOWLEDGEMENTS
We wish to thank Mrs. Eija Myöhänen, Mrs. Helena Kemiläinen and Mrs. Aija Parkkinen
for their skillful technical assistance.
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Table 1. Significant associations between the NRF2 and SRXN1 genotypes and risk of breast cancer. Associated Homozygousb Allele positivityc SNP P(�2)a P(Trend) allele P OR (CI) P OR (CI) NRF2 rs13035806 0.065 0.824 G 0.058 0.353 (0.115-1.085) 0.048 0.339 (0.111-1.040) rs2706110 0.029 0.058 A 0.011 2.079 (1.175-3.679) 0.300 1.162 (0.875-1.543) G 0.011 0.481 (0.272-0.851) 0.010 0.487 (0.279-0.851) rs10183914 0.474 0.913 ns rs1962142 0.335 0.224 ns rs1806649 0.850 0.571 ns rs2364722 0.276 0.348 ns rs6706649 0.141 0.398 ns rs6721961 0.028 0.113 T 0.008 4.656 (1.350-16.063) 0.377 1.150 (0.843-1.569) G 0.008 0.215 (0.062-0.741) 0.008 0.217 (0.063-0.746) rs2886162 0.352 0.449 ns SRXN1 rs6116929 1 0.991 ns rs6076869 0.948 0.922 ns rs6053666 0.052 0.028 C 0.079 0.673 (0.432-1.048) 0.017 0.702 (0.524-0.939) T 0.079 1.486 (0.954-2.314) 0.345 1.215 (0.811-1.820) rs7269823 0.319 0.150 ns rs2008022 0.222 0.364 ns rs13043781 0.535 0.307 ns rs6085283 0.247 0.784 ns rs6053728 0.472 0.257 ns a P from the chisquared test for overall association with breast cancer risk. P(Trend); P value from the Armitage trend test for the overall association with breast cancer risk. ns; no significant association observed. b P, OR and CI for the homozygous allele carriers. c P, OR and CI for the homozygous and heterozygous allele carriers.
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Table 2. Significant associations of the NRF2 and SRXN1 genotypes with cytoplasmic NRF2 and sulfiredoxin protein expression. Allele positivityc Allele positivityc Allele positivityc Associated High extent NRF2 Low extent NRF2 Negative sulfiredoxin SNP P(Trend)a P(Trend) b allele P OR (CI) P OR (CI) P OR (CI) NRF2 rs1962142 0.030 T 0.036 1.742 (1.035-2.933) 0.042 1.990 (1.015-3.901) C 0.230 0.469 (0.133-1.659) 0.086 0.159 (0.009-2.745)
rs6721961 0.0008 T 0.0003 2.420 (1.491-3.926) 0.047 1.867 (1.002-3.478) G 0.274 0.564 (0.199-1.595) 0.042 0.114 (0.007-1.940)
rs2886162 0.041 A 0.011 1.988 (1.162-3.400) G 0.428 0.808 (0.476-1.370) SRXN1 rs6076869 0.012 T 0.005 1.927 (1.217-3.051) A 0.360 0.712 (0.343-1.478)
a P from the Armitage trend test for the overall association with cytoplasmic NRF2 protein expression. b P value from the Armitage trend test for the overall association with sulfiredoxin protein expression. ns; no significant association observed. c P, OR and CI for the homozygous and heterozygous allele carriers.
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Table 3. Variables significantly associated with breast cancer survival in multivariate analysis according to NRF2 SNP genotypes. Variable n B (SE) Wald HR (95% CI) P value
Nodal status Negative 168 Ref.
Positive 122 0.859 (0.235) 13.424 2.362 (1.491-3.740) 0.0002485
HER2 status Negative 251 Ref. Positive 39 0.932 (0.262) 12.633 2.539 (1.519-4.244) 0.000379
rs2886162
GG+GA 219 Ref. AA 71 0.523 (0.243) 4.613 1.687 (1.047-2.718) 0.032
Analysis stratified by tumor grade, nodal status, ER status, PR status, histological type, tumor size, HER2 status, cytoplasmic NRF2 expression and rs2886162 genotypes. HR (95% CI); Hazard ratio of breast cancer death and 95% confidence interval from Cox regression survival analysis. Ref.; Reference category
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FIGURE LEGENDS
Figure 1. Immunohistochemical staining of NRF2 and sulfiredoxin. A) Ductal breast
carcinoma showing strong nuclear positivity for NRF2. B) Ductal breast carcinoma
showing negative nuclear staining for NRF2. C) Ductal breast carcinoma showing strong
cytoplasmic positivity for NRF2. D) Ductal breast carcinoma showing negative cytoplasmic
staining for NRF2. E) Ductal breast carcinoma showing strong cytoplasmic positivity for
sulfiredoxin. F) Ductal breast carcinoma showing negative cytoplasmic staining for
sulfiredoxin. Magnification 250x in A), C) and E), and 110x in B), D) and F).
Figure 2. Association of NRF2 rs2886162 with breast cancer survival in multivariate
analysis. Tumor grade, nodal status, ER status, PR status, histological type, tumor size,
HER2 status, cytoplasmic NRF2 expression and rs2886162 genotypes included in
analysis. HR (95% CI)= Hazard ratio of breast cancer death with 95% confidence interval
in Cox regression analysis.
Figure 3. Association of NRF2 rs2886162 with survival among breast cancer patients with
different therapies. A) Breast cancer specific survival among patients treated with adjuvant
chemotherapy. Analysis stratified by age, stage and radiation therapy. B) Recurrence-free
survival among patients treated with postoperative radiation therapy. Analysis stratified by
age, stage, hormone therapy and chemotherapy. HR (95% CI)= Hazard ratio of breast
cancer death with 95% confidence interval in Cox regression survival analysis.
Figure 4. Significant associations of SRXN1 SNP genotypes with survival among breast
cancer patients receiving radiation treatment. Breast cancer specific survival according to
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A) rs6116929, B) rs2008022, C) rs7269823, and D) rs6085283 genotypes. Analyses
stratified by age, stage, hormone therapy and chemotherapy. HR (95% CI)= Hazard ratio
of breast cancer death with 95% confidence interval in Cox regression survival analysis.
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B
Figure 1.
A B
C D
E F
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Figure 2.
NRF2 rs2886162
GG&GAn=219
P=0.032
AAn=71HR=1.687 (95% CI, 1.047-2.718)
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Figure 3.
A B
GG&GA n=63 GG&GA
n=194
AA n=53HR= 1.68 (95% CI, 1.07-2.64)
AA n=16HR= 2.43 (95% CI, 1.16-5.08)P=0.019
( , )
P=0.025
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A B
Figure 4.
GG
rs6116929 rs2008022
AA&AGn=185
n=64
P 0 049
CA&AAn=107
CCn=185HR=1.74 (95% CI 1.00-3.00)
P=0.049P=0.020
CCn=134HR=1.73 (95% CI 1.09-2.74)
C Drs7269823C rs6085283
CC
D
AG&GG
AAn=148
P=0.045 CT&TT170
CCn=76
n=100HR=1.53(95% CI 1.01-2.41)
P=0.036n=170HR=1.75 (95% CI 1.04-2.95)
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-12-1474
Published OnlineFirst September 10, 2012.Cancer Res Jaana M. Hartikainen, Maria Tengström, Veli-Matti Kosma, et al. sulfiredoxin predict survival outcomes in breast cancerGenetic polymorphisms and protein expression of NRF2 and
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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 10, 2012; DOI: 10.1158/0008-5472.CAN-12-1474